This invention relates generally to antigenic preparations and specifically to four Leptospira membrane proteins i.e., kinase, permease, mannosyltransferase and endoflagellin which are used to induce a protective immune response in animals. Such proteins can be used immunologically as vaccines for leptospirosis caused by this organism. Alternatively, diagnosis of leptospirosis can be performed by detecting the presence of these proteins, antibody to the proteins, or polynucleotides which encode the proteins.
The pathogenic species of the Leptospira genus are the causative agents of leptospirosis, a zoonotic disease of worldwide importance. The bacterium is a gram negative spirochete which thrives under aerobic conditions. These bacteria have fastidious nutritional requirements and are able to utilize long chain fatty acids as a sole source of carbon. Leptospires are motile and their rapid, corkscrew motility serves as a distinguishing identifying feature of this organism. Leptospires are resistant to both metronidazole and 5-fluorouracil, and demonstrate a generation time of 10 to 12 hours in vitro.
All pathogenic Leptospires were formerly classified as Leptospira interrogans. Recently DNA homology studies have led to the reclassification of Leptospira interrogans into seven pathogenic Leptospira species: L. borgpetersenii sv hardjobovis, L. inadai, L. interrogans, L. kirshneri, L. noguchii, L. santarosai and L. weilli. The serology of pathogenic Leptospira species which are responsible for Leptospirosis disease indicates that there are more than two hundred serovars (xe2x80x9csvxe2x80x9d) within twenty three serogroups (Farr, R. W. Clin. Infect. Dis. 21:1-8 (1995)). There are many serovars responsible for leptospirosis disease worldwide. Leptospira interrogans sv pomona is a common isolate from infected swine where it causes fever, jaundice, hemoglobinuria, and renal failure. Leptospira interrogans sv hardjobovis is an important cause of bovine disease, where it causes abortion and agalactia, and poses a zoonotic threat to humans after prolonged exposure to infected cattle. Many infected animal are asymptomatic. These animals can however act as carriers and shed leptospires through urine.
Leptospira infections in cattle demonstrate a 16% infection rate, with rates being higher in beef cattle than in dairy cattle. The infection rate is also higher in bulls than in cows. There is also a marked prevalence of certain serovars, which cause bovine leptospirosis. In a recent survey of cattle in the 48 states in the USA, of those animals positive for leptospira, 84% were infected with sv hardiobovis, 12% were infected with sv pomona, and 4% were infected with sv griptotyphosa (Miller, D. A. et al., Am. J. Vet. res. 52(11):1761-1765 (1991)).
Little is known concerning the pathogenesis of Leptospira infections. Infections are usually transmitted by contact with urine from an infected animal. Soil and water which has been contaminated with infected urine can also transmit infection, although the prolonged survivability of leptospires under these conditions is questionable. Survival of Leptospira outside the host is fostered by a temperature of 22xc2x0 C. or above, moisture, and a neutral to slightly alkaline environment. Leptospira are readily killed by temperatures above 60xc2x0 C., detergents, desiccation, and acidity. Once the leptospira has invaded the host, attachment to and penetration of the intercellular junctions of mucosal epithelial cells is a crucial step in the infection. A bacteremia usually results and is the first pathological condition associated with leptospirosis. Once in circulation, the bacterium can colonize the kidneys, where they may cause an acute or chronic infection. Some potential virulence factors involved in the pathogenesis of leptospira are, hyaluronidase, urease, haemolysins, and phospholipases.
Leptospirosis is caused by pathogenic strains of Leptospira which are capable of infecting most mammalian species. The predominant natural reservoirs of pathogenic Leptospira are wild mammals, although other vertebrates occasionally are infected. In addition, several species of leptospires are known pathogens of marine mammals, including Pacific harbor seals (Stampler, M. A. et al., J. Wild. Dis. 34(2):407-410 (1989)). Domestic animals such as dogs, cattle, swine, sheep, goats and horses, also may be major sources of human infections. Infection occurs either through direct contact with an infected animal or indirect contact with contaminated soil or water. In livestock, the disease causes economic losses due to abortion, stillbirth, infertility, decreased milk production, and death.
The severity of human leprospirosis varies greatly and is determined to a large extent by the infecting strain and by the general health of the host. The improved ability of regional laboratories to group Leptospira has resulted in the recognition of the large number of serovars endemic in the United States, as well as the extent of infections in a variety of animal species. Nevertheless, it is an infrequently diagnosed human disease. Approximately 100 cases are reported annually in the United States.
Because of its prevalence in rodents and domestic animals, leptospirosis has been primarily a disease of person in occupations heavily exposed to animals and animal products, such as sewer workers, swineherders, veterinarians, abattoir workers, and farmers (Vinetz, J. M., Cur. Opin. Infect. Dis. 10:357-361 (1997)). Also at risk are persons living in rodent-infested housing, such as urban slums, and dog owners. There is a higher incidence in males. At present, the majority of cases occur in the summer and fall in teenagers and young adults. Avocational exposure is now increasingly common.
Common source outbreaks attributed to contaminated ponds or slowly moving streams are numerous. A high attack rate, summer season, young age group, and the proximity of animals to the water typify most of these outbreaks. In some areas of the world, the runoff during flooding also is highly infectious.
Sporadic disease may be acquired by direct contact with infected animals. Vaccination of domestic animals, which prevents clinical disease, may fail to prevent shedding of Leptospira. Pet dogs have been a prominent source of sporadic human cases. The convoluted renal tubules of animal reservoirs harbor viable Leptospira, which are passed in the urine. The duration of asymptomatic urinary shedding varies with the animal species; humans rarely shed Leptospira longer than a few months.
Forms of transmission other than direct and indirect contact with contaminated urine are rare. Lactating animals shed Leptospira in the milk, but whole milk is leptospirocidal after a few hours, and no known human cases have occurred in this manner. Leptospira are not shed in saliva, and animal bites are therefore not a direct source of infection.
The pathogenesis of leptospirosis is very similar to that of other spirochetal diseases, including syphilis (caused by Treponema pallidum) and Lymeborreliosis (caused by Borrelia burgdoferi). Both syphilis and Lyme borreliosis are are characterized by widespread dissemination early in the course of disease, including invasion of the central nervous system. Leptospira share this ability with other pathogenic spirochetes such that meningitis is a common manifestation of leptospirosis. Another feature of spirochetal infections is the ability to persist chronically in the host, as manifested in cases of tertiary syphilis and chronic Lyme arthritis. (For a comprehensive review, see Baranton, G. and Old, I. G., Bull. Inst. Pasteur 93:63-95 (1995)).
Efforts to control leptospirosis have been hampered because virulent leptospires have the capacity for both long-term survival in the environment as well as persistent infection and shedding by wildlife and livestock.
Leptospira membrane proteins are of great importance because they play a key role in bacterial pathogenesis. The identification of membrane proteins involved in Leptospira pathogenesis is significant to understanding not only leptospiral membrane proteins and their involvement in pathogenesis, but also to understanding other spirochetal membrane proteins and their role in pathogenesis.
Currently available leptospiral vaccines produce short-term immunity and do not provide cross-protection against many of the 170 serovars of pathogenic Leptospira (Thiermann, et al., J. Am. Vet. Med. Assoc. 184:722 (1984)). These vaccines consist of inactivated whole organisms or outer envelope preparations which produce seroreactivity as determined by microscopic agglutination of intact organisms. The nature of the protective immunogens in these vaccine preparations has not been conclusively elucidated, although several lines of evidence suggest that lipopolysaccharide-like substance (LLS) may confer a degree of protection.
In terms of treatment of active infection, oxytetracycline is the drug of choice and is used routinely in the field to both cure infection and carriage. Several vaccine preparations using bacterins or components of lipopolysaccharide have been used with variable success. Protection with the current vaccines tend to be serovar specific and lack the ability to generate a reproducible degree of protection.
The present invention is based on the identification of four Leptospira membrane proteins i.e., kinase, permease, mannosyltransferase and endoflagellin, which are associated with pathogenic strains of Leptospira. Due to spirochetal membrane fragility and the fact that membrane proteins are present in small amounts, there have been limited definitive reports of membrane spanning spirochetal membrane proteins until the present invention. The identification of in vivo expressed genes by mRNA subtractive hybridization is a powerful means by which to identify virulence-related genes. The present invention describes the identification of three Leptospira interrogans sv pomona genes which are expressed during colonization of the liver of infected Syrian hamsters. The present invention also describes a fourth gene identified from L. hardjobovis using a ZAP expression library. The invention also describes four membrane proteins from Leptospira which are immunogenic and useful for inducing an immune response to pathogenic Leptospira as well as providing a diagnostic target for leptospirosis.